This invention relates to biologically compatible material for use in transplants, and to the production and use of such material.
The replacement of failed or faulty animal (particularly human) tissue, including organs, has over the last four decades become a common place therapy in clinical medicine. These replacement therapies range for example from the use of the polyethylene terephthalate sold under the trade mark DACRON by DuPont to repair faulty blood vessels to the use of saphenous vein as an autograft to by-pass blocked arteries and to the transplantation from one human to another of a heart.
Organ transplantation has undergone significant development with modern immunosuppressants allowing high success rates to be achieved at relatively modest cost. The demand for organ transplantation has increased rapidly. There are now more than 20,000 organ transplants per annum carried out worldwide. This, however, represents only approximately 15% of the need as assessed by current criteria. The supply/demand ratio of donor organs of all types can not be met from existing sources. This is perhaps best illustrated with the demand for heart transplantation. The first heart transplantation by Barnard in 1967 generated considerable press coverage. Within a year, 101 heart transplants had been performed in 22 countries by 64 different surgical teams. Disillusionment followed the poor results obtained so that by the early 1970s fewer than 30 transplants per year were being performed worldwide. The introduction of cyclosporin immuno-suppression, however, has revolutionarised heart transplantation so that most centres can now anticipate success rates for heart transplantation of more than 80% one year graft (and patient survival). As expertise is gained, this survival rate can reasonably be expected to increase further. The success of this procedure, of course, fuels demand so that the medical profession and the general public become more aware that heart transplantation offers a real alternative to death, so more and more patients are referred for the procedure. Currently, over 2,000 heart transplants per annum are performed.
Today, the greatest risk of death in heart transplantation is while waiting for a suitable donor organ to become available. While the artificial heart offers a short-term support device for these patients, long-term demands are for more heart transplant centres and a greater donor supply. The potential number of individuals who might benefit from cardiac transplantation has never been scientifically established, but published estimates of the need for heart transplantation have ranged widely between 50 and 250 people per million per year depending on selection criteria, age of recipient, disease and so forth. Whatever the actual figure may be, it is quite clear already that current donor supply options are incapable of meeting demand. Similar comments can be made for kidney and liver transplantation, and it seems likely that once pancreas or Islet of Langerhans cell transplantation becomes a widely-accepted therapeutic procedure for the treatment of diabetes, shortage of this tissue will also become a prime concern.
There are further disadvantages with current transplantation therapy. It is by no means always the case that donor organs are fit for use in transplantation, not least because many organ donors are themselves victims of some accident (for example, a road accident) which has caused death by injury to some organ other than that which is being transplanted; however, there may be some additional injury to or associated difficulty with the organ to be transplanted.
Further, because of the unpredictable availability of organs from donors, transplant surgery often can not be scheduled as a routine operation involving theatre time booked some while in advance. All too frequently, surgical teams and hospital administrators have to react the moment a donor organ is identified and work unsocial hours, thereby adding to administrative and personal difficulties.
In the case of heart, liver and lung transplants, if rejection is encountered it will not usually be possible to retransplant unless by chance another suitable donor becomes available within a short space of time.
Apart from the above medical difficulties, current transplantation practice can in some cases involve social difficulties. In the first place, there may be religious objections to removing organs from potential donors, particularly in cultures believing in reincarnation. There are of course other ethical and social difficulties encountered in removing organs from dead humans, particularly as consent is required in some countries. Finally, the appearance of a commercial trade in live kidney donors is causing concern, particularly in certain third world countries, and it would be socially desirable to suppress or reduce such a trade.
Conventional transplantation surgery, as outlined above with its disadvantages, involves the transplantation from one animal of a particular species (generally human) to another of the same species. Such transplantations are termed allografts. Because of the difficulties with conventional allograft supply, as outlined above, attention has focused on the possibility of using xenografts in transplantation. Xenografting is the generic term commonly used for the implantation of tissues, including cells and organs, across species barriers.
There have already been several examples of the successful use of xenografts in therapeutic replacement schedules. For example, recent years have witnessed the use of pig tissue for aortic valve replacement, pig skin to cover patients with severe burns, and cow umbilical vein as a replacement vein graft. All of these xenografts have, however, one point in common: they provide a mechanical replacement only. The tissue used is biologically non-functional. The reason for this is that the immune processes existing in man immediately (within minutes or hours) destroy the cellular integrity of tissues from most species. Such xenografts are known as discordant xenografts.
The ferocity of this destruction is phylogenetically associated. Thus, tissue from the chimpanzee, which is a primate closely related to man, can survive in man in much the same way as an allograft; such a xenograft is known as a concordant xenograft.
While it may be thought that concordant xenografts might provide the answer to the difficulties with allografts, in practice this is probably not the case. Chimpanzees are much smaller than man and chimpanzee organs are generally not big enough to work in man. In the case of kidneys this may be overcome by transplanting two chimpanzee kidneys to replace a failed human kidneys, but for liver and heart this is clearly not a possibility. Furthermore, chimpanzees breed slowly in nature and poorly in captivity, and the demand for chimpanzees as experimental animals (particularly in the current era of research into Acquired Immune Deficiency Syndrome (AIDS)) means that, yet again, demand is outstripping supply. Additionally, there may be some social difficulty with the public acceptance of the use of other primates as xenograft donors.
Attention has therefore refocused on discordant xenografts. It has been commonly believed that the reason why discordant xenografts fail so rapidly, is the existence in the recipient species of xe2x80x9cnaturally occurringxe2x80x9d antibodies against as yet undefined antigens of the donor species (Shons et al (Europ. Surg. Res. 5 26-36 (1973)). The antibodies are called xe2x80x9cnaturally occurringxe2x80x9d because they are found to exist in individuals who have not had any immunological challenge from the donor species.
The rapid rejectionxe2x80x94known as hyperacute antibody-mediated rejectionxe2x80x94of an organ graft is well documented. In the early 1960s, when (allograft) kidney transplantation became a routine treatment, it was observed that transplanted kidneys were occasionally rejected by the recipient whilst the operation was still in progress. During a transplant operation, the kidney will as a rule become red and firm in consistency soon after the vessels of the recipient and donor are sutured together. Such transplants often produce urine almost immediately. In the form of rejection where the graft is destroyed while the patient is still on the table (hyperacute rejection) the destructive processes begin in the first few minutes or so after transplantation. When this occurs, the kidney becomes bluish and patchy and then congested. The consistency of the organ is also altered. As a rule, the graft becomes oedematous, no urine production occurs and the newly-transplanted kidney is then immediately removed. It has become clear that a humorally-mediated immunological response between preformed circulating antibodies in the recipient and antigens in the donor kidney are involved. The only way to avoid its occurrence in allografting is to check before transplantation that there are no antibodies existing in the recipient against the donor cells. With increased knowledge of testing for such antibodies (known as the cross match) it has become clear that this generalisation that antibody in the recipient reacts against antigens in the donor is not true and that hyperacute graft destruction, when it involves transplants between individuals of the same species is restricted to the existence of specific sorts of antibody known as T-warm positive cross-match: and almost certainly these antibodies belong to the IgG subclass. Furthermore, the presence of these antibodies always results from a pre-existing immunisation procedure either as a result of previous blood transfusions or as a result of pregnancy or, most commonly, as a result of a failed previous transplant.
The mechanism for hyperacute xenograft rejection has largely been thought to be much the same as the mechanism for hyperacute allograft rejection, as outlined above. The literature on the mechanism of xenograft rejection is extensive, stretching back some 83 years. During that time, only three publications appear to have suggested a mechanism for xenograft rejection which does not involve antibodies. The suggestion was that the alternative pathway of complement activation was implicated in xenograft rejection (although not necessarily using such terminology). The suggestion first appeared in 1976 in a paper by Schilling et al (Surgery. Gynaecology and Obstetrics 142 29-32 (1976)). The suggestion was made again in 1988 and 1989 (the same data were published twice) by Miyagawa et al (Transplantation 46(6) 825-830 (1988) and Transplantation Proceedings 21(1) 520-521 (1989)). However, the results were not conclusive, because both these experiments suffered from substantially the same fault. The model chosen is claimed by the authors to be a xenograft model in which cross-species antibodies did not exist. However, it now appears that the assays used to detect cross-species antibodies were inadequate, and that the inferences drawn in these papers were based on inadequate data.
Most measures currently taken experimentally to avoid or reduce rejection in xenografts involve chemotherapeutically interfering with the recipient""s immune system, largely on a non-specific basis for example with cyclosporin A and other immunosuppressants, by plasmaphoreses, by treatment with cobra venom factor, Staphylococcus protein A absorption of antibody and so on. This approach naturally follows from the chemotherapy that supports allografts.
This invention adopts a radically different approach: instead of non-specifically interfering with the recipient""s immune system, the invention enables to co-administration of material which has the effect of the donor graft being regarded as self by certain components of the recipient""s immune system. In particularly preferred embodiments, the donor tissue itself is modified to appear immunologically to the recipient to be self in certain respects.
It is has also been discovered that hyperacute xenograft rejection is not necessarily antibody-mediated. This arises from two observations. First, in the absence of antibody but the presence of complement, hyperacute rejection is observed; secondly, in the presence of antibody but the absence of complement, no hyperacute rejection is observed.
The invention is based on the discovery that complement activation is pre-eminent in the hyperacute destruction of a xenograft whether or not such activation is aided by the binding of appropriate antibody molecules. Activation of the alternative pathway of complement can be induced by a variety of cell products. These products are not restricted to foreign-invading cells such as bacteria or xenografts but exist on many cells. Thus, in principle, many cells of an individual could activate the alternative pathway of complement, causing massive auto-immune destruction. That this does not happen is due to the existence of a number of complement down-regulating proteins naturally present in serum and on the surface of cells. These molecules (referred to herein as xe2x80x9chomologous complement restriction factorsxe2x80x9d) prevent the complete activation of self complement either by the classical or alternative pathway by the products of self cells, thus preventing the auto-immune destruction of self. The functioning of such molecules is elegantly illustrated in paroxysmal nocturnal haemoglobinuria. In this disease, the membrane anchor of at least one of these molecules (decay accelerating factor) is absent. Thus, the protein is not retained in the erythrocyte cell membrane and detaches from the cell, which activates the alternative pathway of complement and is then lysed thus causing haemoglobinuria.
According to a first aspect of the present invention, there is provided a method of transplanting animal tissue into a recipient, wherein the tissue is derived from a donor of a different species from the recipient, the donor species being a discordant species with respect to the recipient, the method comprising grafting the tissue into the recipient and providing in association with the grafted tissue one or more homologous complement restriction factors active in the recipient species to prevent the complete activation of complement.
The word xe2x80x9ctissuexe2x80x9d as used in this specification means any biological material that is capable of being transplanted and includes organs (especially the internal vital organs such the heart, lung, liver and kidney, pancreas and thyroid) cornea, skin, blood vessels and other connective tissue, cells including blood and haematopoietic cells, Islets of Langerhans, brain cells and cells from endocrine and other organs and body fluids (such as PPF), all of which may be candidates for transplantation from one species to another.
A xe2x80x9cdiscordant speciesxe2x80x9d is a species a (generally vascularised) xenograft from which into the recipient would normally give rise to a hyperacute rejection, that is to say rejection within minutes or hours and not days (Calne Transplant Proc 2:550, 1970). Such hyperacute rejections will be well known to those skilled in the art, and ay take place in under 24 hours, under 6 hours or even under one hour after transplantation.
Complement and its activation are now well known, and are described in Roitt, Essential Immunology (Fifth Edition, 1984) Blackwell Scientific Publications, Oxford. The activity ascribed to complement (Cxe2x80x2) depends upon the operation of nine protein components (C1 to C9) acting in sequence, of which the first consists of three major sub-fractions termed Clq, Clr and Cls. Complement can be activated by the classical or alternative pathway, both of which will now be briefly described.
In the classical pathway, antibody binds to C1, whose Cls subunit acquires esterase activity and brings about the activation and transfer to sites on the membrane or immune complex of first C4 and then C2. This complex has xe2x80x9cC3-convertasexe2x80x9d activity and splits C3 in solution to produce a small peptide fragment C3a and a residual molecule C3b, which have quite distinct functions. C3a has anaphylatoxin activity and plays no further part in the complement amplification cascade. C3b is membrane bound and can cause immune adherence of the antigen-antibody-C3b complex, so facilitating subsequent phagocytosis.
In the alternative pathway, the C3 convertase activity is performed by C3bB, whose activation can be triggered by extrinsic agents, in particular microbial polysaccharides such as endotoxin, acting independently of antibody. The convertase is formed by the action of Factor D on a complex of C3b and Factor B. This forms a positive feedback loop, in which the product of C3 breakdown (C3b) helps form more of the cleavage enzyme.
In both the classical and alternative pathways, the C3b level is maintained by the action of a C3b inactivator (Factor I). C3b readily combines with Factor H to form a complex which is broken down by Factor I and loses its haemolytic and immune adherence properties.
The classical and alternative pathways are common after the C3 stage. C5 is split to give C5a and C5b fragments. C5a has anaphylatoxin activity and gives rise to chemotaxis of polymorphs. C5b binds as a complex with C6 and C7 to form a thermostable site on the membrane which recruits the final components C8 and C9 to generate the membrane attack complex (MAC). This is an annular structure inserted into the membrane and projecting from it, which forms a transmembrane channel fully permeable to electrolytes and water. Due to the high internal colloid osmotic pressure, there is a net influx of sodium ions and water, leading to cell lysis.
Homologous complement restriction factors (HCRFs) useful in the present invention can in general interfere with any part of the complement activation cascade. An HCRF may interfere solely with that part which constitutes the classical pathway, or solely with that part which constitutes the alternative pathway, or more usually may interfere with that part which is common to both the classical and alternative pathways. It is preferred that the HCRF regulator interfere with the common part of the pathway. The HCRF may be identical to a natural HCRF or simply have the appropriate function. Synthetic and semi-synthetic HCRFs, including those prepared by recombinant DNA technology and variants however prepared, are included within the term HCRF.
As has been mentioned above, homologous complement restriction factors are substances which regulate the action of the complement cascade in such a way as to reduce or prevent its lytic activity; they are used by the animal body to label tissue as self to avoid autoimmune reaction. In this invention it is possible in principle for the HCRF to be either membrane bound or free in serum, although in practice it will be preferred to have the HCRF being membrane bound on cells of the xenograft tissue. In this way, it is easier for the HCRF to be xe2x80x9cin association withxe2x80x9d the graft tissue. Preferred HCRFs include putative cell membrane factors including the C3b/C4b receptor (CR1), C3. dg receptor (CR2), decay accelerating factor (DAF), C3b Inactivator and membrane cofactor protein (MCP). Putative serum HCRFs include Factor H, decay accelerating factor (DAF) and C4 binding protein (C4bp). These HCRFs all down-regulate the activity of complement by interference at the C3 stage. Homologous restriction factor (HRF), which blocks at C8, is also a putative membrane factor.
Many, but not all, of the genes for suitable HCRFs are located in the RCA (regulator of complement activation) locus, which map to band q32 of chromosome 1 (Rey-Campos et al J. Exp. Med. 167 664-669 (1988)).
Although there has been some confusion with the nomenclature and location of HCRFs, the factors C4BP, CR1, DAF and Factor H are identified by Rey Campus et al (loc. cit.) and in their earlier study (J. Exp. Med. 166 246-252 (1987)). Membrane cofactor protein (MCP) is treated by some workers as synonymous with C4 binding protein (C4bp) and it may be that these two factors are either related or identical. Rother and Till (xe2x80x9cThe Complement Systemxe2x80x9d, Springer-Verlog, Berlin (1988)) review the regulatory factors of C3 convertase in section 1.2.3.2; they equate C4 binding protein (C4bp) with decay accelerating factor and Factor H with B1H-protein and C3b Inactivator Accelerator. No doubt the nomenclature, localisation and characterisation of HCRFs will continue to evolve, but it is to be understood that the present invention contemplates the use of all HCRFs as suitability and preference dictate.
Other references to HCRFs are as follows:
Factor I (also previously known as C3b inactivator or KAF):
Tamura and Nelson (J. Immunol. 99 582-589 (1967);
Factor H: Pangburn et al (J. Exp. Med. 146 257-270 (1977);
C4 binding protein: Fujita et al (J. Exp. Med. 148 1044-1051 (1978));
DAF (also known as CD55): Nicholson-Weller et al (J. Immunol. 129 184 (1982));
Membrane Cofactor Protein (MCP; also known as CD46 and first described as gp45-70 and further known as gp66/56): Seya et al (J. Exp. Med. 163 837-855 (1986));
CR1 (also known as CD35): Medof et al, (J. Exp. Med. 156 1739-1754 (1982)) and Ross et Al (J. Immunol. 129 2051-2060 (1982));
CR2 (also known as CD21, 3d/EBV receptor and p140): Iida et al (J. Exp. Med. 158 1021-1033 (1983)) and Weis et al (PNAS 81 881-885 (1984)).
The tissue distribution of some of the RCA proteins are as follows:
CR1: Membrane (limited): erythrocytes; monocytes; most B and some T cells; polymorphonuclear leukocytes; follicular-dendritic cells; glomerular podocytes;
CR2: Membrane (limited): most B cells; follicular-dendritic cells; some epithelial cells and a few T cell lines;
MCP: Membrane (wide): all peripheral blood cells (but erythrocytes); epithelial, endothelial and fibroblast cell lineages; trophoblast and sperm;
DAF: Membrane (wide): all peripheral blood cells; epithelial, endothelial and fibroblast cell lineages; trophoblast and sperm;
C4bp: Plasma: liver synthesis; and
H: Plasma: liver synthesis; fibroblast and monocyte cell lines.
As for proteins involved in homologous restriction at the level of the membrane attack complex, the use of which is also contemplated by means of the present invention, there is general agreement (but as yet no proof) in the form of a protein sequence that the following 65kDa (or thereabouts) proteins are identical:
C8 binding proteins (Schonermark et al, J. Immunol. 136 1772 (1986));
homologous restriction factor (HRF) (Zalman et al Immunology 83 6975 (1986)); and
MAC-inhibiting protein (MIP) (Watts et al. (1988)).
The C8bp/HRF/MIP protein is attached to the cell surface by means of a glycolipid anchor, as are CD59 and DAF: these proteins are known to be functionally absent in paroxysmal nocturnal haemoglobinuria.
An 18-20 kDa protein is also implicated at the MAC level. The following are believed to be identical (but may not be):
P-18 (Sugita et al (J. Biochem 104 633 (1988)));
HRF-20 (Okada et al (Intl. Immunol 1 (1989)));
CO59 (Davies et a (J. Exp. Med. (September 1989))); and
Membrane inhibitor of reactive lysis (MIRL) (Hologuin et al J. Clin. Invest 84 7 (1989))).
The evidence for the putative identity of these proteins is that the protein and/or CDNA sequences for CD59 and HRF-20 are shown to be identical: probably they are the same as P-18/MIRL also. It should be noted that there is some homology of the CD59/HRF.20 sequence with that of murine LY-6 antigen, which is involved in T-cell activation (Gronx et al (J. Immunol. 142 3013 (1989))).
SP-40.40 is also involved in MAC regulation (Kivszbaum et al EMBO 8, 711 (1989)).
It is preferred that the HCRF interfere with complement activation at the C3 stage. MCP and DAF both block the positive feedback loop in the alternative pathway of C3 activation, and these constitute preferred HCRFs.
The HCRF is provided in association with the grafted tissue. This means that the HCRF is administered in such a way that the graft tissue is labelled as self, but other foreign material, such as invading bacteria, are not significantly so labelled. It may be possible simply to administer parenterally, but locally to the graft tissue, one or more appropriate HCRFS. However, in practice this may not be preferred because of the difficulty of causing adequate localisation of the HCRF at the graft tissue and because of the further difficulty that the HCRF may have to be repeatedly administered to the recipient after the graft has taken place; however, this could be overcome by the use of specialist pharmaceutical delivery systems.
It will generally be much more convenient to provide the HCRF in such a way that it is integrated with the cell membrane on donor tissue. Although there may be some benign infections of the transplanted tissue which could cause suitable expression, by far the most preferred route of achieving this end is for the donor tissue to be transgenic in that it contains and expresses nucleic acid coding for one or more HCRFs active in the recipient species when grafted into the recipient. Such transgenic tissue may continue to express an HCRF indefinitely. The HCRF may be genetically derived from the recipient species or less preferably from a closely related species for which concordant xenografts may be possible.
Although in principle the transgenic donor tissue may come from a cell culture, it is preferable for the donor tissue to come from a transgenic animal. The transgenic animal should express (or be capable of expressing) the HCRF in at least the tissue to be transplanted, for preference. However, even this is not essential, as it may be possible to bind the HCRF to the cell membranes of the donor tissue by some binding agent (such as a hybrid monoclonal antibody (Milstein and Cuello Nature 305 537 (1983)) or receptor.
The recipient species will primarily be human, but not exclusively. Other primates may be suitable recipients, as may any other species where the economics and ethics permit.
The donor species may be any suitable species which is different from the recipient species and which, having regard to the physiology of the recipient species is able to provide appropriate tissue for transplantation. For human recipients, it is envisaged that pig donors will be suitable, but any other species may be suitable.
According to a second aspect of the invention, there is provided graftable animal cells or tissue of a donor species, the cells or tissue being associated with one or more homologous complement restriction factors active in the intended recipient species to prevent the complete activation of complement, the donor species being a discordant species with respect to the recipient species.
According to a third aspect of the invention there is provided a transgenic animal having transplantable tissue, which does not give rise to xenograft rejection on transplantation into or exposure to the immuno system of at least one discordant species. A discordant species is one which would normally hyperacutely reject a xenograft from the animal.
The invention therefore encompasses the use of animal tissue derived from a donor species and one or more homologous complement restriction factors active in a recipient species, wherein the donor species is a discordant species in relation to the recipient species, in the preparation of tissue graftable into the recipient species.
According to a fourth aspect of the invention, there is provided a transgenic animal having cells capable of expressing a homologous complement restriction factor of another species. The homologous complement restriction factor will generally be active in a species which is discordant with respect to the species of the transgenic animal. The cells may be of one particular tissue, with preferences being as described with reference to the first aspect of the invention, or of more than one or all tissues, in which case the animal may become a donor of more than one tissue. Such a transgenic animal may be regarded as a collection of non-transformed (in the sense of non-proliferative) cells.
According to a fifth aspect of the invention, there is provided a non-transformed animal cell capable of expressing one or more homologous complement restriction factors active in a species which is discordant with respect to the animal cell.
According to a sixth aspect of the invention, there is provided recombinant DNA comprising DNA coding for at least one homologous complement restriction factor and one or more sequences to enable the coding DNA to be expressed by a non-transformed animal cell. The animal cell may be a cell of a transgenic animal genetically incorporating the construct. As an alternative, the cell may be a cultured organ or other tissue such as an Islet of Langerhans.
According to a seventh aspect of the invention, there is provided a genetic construct suitable for incorporation into the genetic material of an animal to produce a transgenic animal, the construct comprising DNA coding for at least one homologous complement restriction factor and one or more sequences to enable the coding DNA to be expressed in at least some cells of a transgenic animal genetically incorporating the construct. Such a genetic construct may be in the form of a mini chromosome known as a YAC. As above, the homologous complement restriction factor will generally be active in a species which is discordant with respect to the species of the transgenic animal.
According to a eighth aspect of the present invention, there is provided a method of preparing a transgenic animal, the method comprising incorporating into an animal""s genetic material DNA coding for at least one homologous complement restriction factor and one or more sequences to enable the coding DNA to be expressed in at least some cells of the transgenic animal.
Methods of producing transgenic animals are in general becoming more widespread, and the detailed steps to be taken may be as now conventionally used in the art. For example, WO-A-8800239 discloses the steps needed in principle to construct a transgenic animal.
The actual method of incorporation of the construct into the cells of the transgenic animal may be by micro-injection, by sperm-mediated incorporation or any other suitable method. The preliminary genetic manipulation may be carried out in a prokaryote, as is generally preferred.
DNA coding for HCRFs is either available in cDNA form or may be deduced using conventional cloning techniques. The DNA coding for decay accelerating. factor (DAF) is probably the best characterised and has been described by Medof et al (PNAS 84 2007-2011 (1987)). A physical map of the RCA gene cluster is given in Rey-Campos et al (1988) (loc. cit.). Variants of DAF and their preparation by recombinant DNA technology are disclosed in EP-A-0244267; such variants may be used in the present invention.
Because of the better characterisation of the genetics of DAF, and the known sequence of cDNA encoding DAF, DAF constitutes a preferred homologous complement restriction factor.
Other preferred features of the second to seventh aspects of the invention are as for the first aspect, mutatis mutandis.